Several studies have shown that recombinant FVIII products often have discrepant potencies when different assay methods are applied. In the case of one recombinant product, use of the chromogenic assay, mandated in Europe, would mean that 30% less FVIII would be delivered to patients on a molar basis than if one applied the standard one-stage clotting assay used in the United States. In contrast, a B-domain deleted recombinant FVIII gave about 1.25 fold higher potency in the one-stage clotting assay than in the chromogenic assay. It is important for us to discern the mechanism responsible for such discrepancies, to achieve standardization of potency measurements for recombinant FVIII. Results: The activity of FVIII from six different recombinant and plasma-derived FVIII preparations were examined with either one-stage clotting or chromogenic assays. We found that one recombinant FVIII product had discrepancy in its activity when assayed by the two different assay methods. The chromogenic assay gave significantly higher activity than the one-stage clotting assay (Chromogenic/one-stage=1.445, n=7, 0.001>p>0.0001). By immunoprecipitation and Western-blotting analysis, we found that this FVVIII had more polypeptide species that contained the B-domain of FVIII compared to plasma-derived FVIII. In addition, we found a >200 kDa band not observed in plasma-derived FVIII. Antibody mapping suggests that the >200 kDa polypeptide is an N-terminal truncated single polypeptide FVIII. Thrombin catalyzes the activation of FVIII by cleaving several peptide bonds located on the heavy and light chains of the FVIII molecule. To evaluate the effect of the B-domain and other acidic regions of FVIII on thrombin-catalyzed activation, we first asked whether there is a complementary binding site(s) on thrombin in addition to the catalytic site for FVIII. a-Thrombin, blocked at its active site by D-Phe-Pro-Arg Chloromethyl Ketone (PPACK), inhibited thrombin activation of FVIII. This suggests that a site(s) on thrombin, other than the active site, is needed for FVIII activation. Thrombin-catalyzed cleavages of both the heavy and light chains of FVIII were inhibited by a sulfated polypeptide segment of the FVIII heavy chain, residues 714-740 (F8II). F8II did not inhibit either the amidolytic or the clotting activity of thrombin. This further supports the involvement of sites on thrombin, other than the active site, in FVIII activation. To investigate the potential importance of the fibrinogen-recognition exosite of thrombin on FVIII cleavage, the C- terminal dodecapeptide of hirudin named Hirugen, which binds to the exosite, was tested as an inhibitor of thrombin proteolysis of FVIII. The peptide only partially inhibited the digestion of FVIII, mainly on the light chain, by a-thrombin. Furthermore, g-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII, at positions similar to those cleaved by a-thrombin. We conclude that a novel recognition site on thrombin, in addition of the fibrinogen- recognition exosite and the active site of thrombin, is involved in activation of FVIII. An acidic region (residues 714-740) of FVIII binds to one of the sites on thrombin involved in the rate-limiting step of the reaction. Future studies: 1) Identify the novel secondary binding site on thrombin for FVIII activation. 2) Characterize the effect of such binding on FVIII activity and the impact of the binding on the discrepancies between the one-stage clotting and chromogenic assays. 3) Identify and characterize the FVIII-binding site(s)on FXa. We will also evaluate the effect of the N-terminal truncated greater than kDA FVIII or other proteins, such as von Willebrand Factor, on the FVIII activity assays.